The objective of this research proposal is to aid in the discovery of a safe and effective, prophylactic HIV vaccine. The proposed structural studies will directly impact on basic HIV vaccine discovery research by revealing key features of the HIV-1 Envelope (Env) proteins, gp120 and gp41, that are conserved and can be targeted by neutralizing antibodies. The Env proteins gp120 and gp140 are the essential targets for design of a neutralizing antibody-based HIV-1 vaccine. However, the neutralization-sensitive epitopes on gp120 and gp41 are either difficult to access or are not immunogenic. What is critically needed at present is an understanding of the arrangement and accessibility of these functionally conserved epitopes on intact Env trimers. Thus, this research will focus on the design and x-ray crystallographic structural investigations of trimeric gp140 constructs in complex with available neutralizing antibodies. In addition, design and structural characterization of an assortment of carbohydrate and peptide antigens in various contexts as complexes with the current arsenal of HIV-1 broadly neutralizing antibodies, 2G12, 4E10, and Z13e1, will be carried out to assess and advance their utility as immunogens and vaccine candidates. Furthermore, crystal structures will be determined for any exciting, new, broadly neutralizing antibodies in complex with their antigens as soon as they are discovered. The overall goal of this work is to uncover the points of vulnerability in the viral gp120 and gp41 proteins, i.e. HIV-1's Achilles' heel, that can be exploited for design of antigens that can elicit an effective protective humoral response from an HIV-1 vaccine. The specific aims are to design and characterize stable HIV-1 trimers to advance their potential use as immunogens, to investigate novel carbohydrate-based antigens as potential immunogens, to advance the utility of the highly conserved membrane proximal external region (MPER) in gp41 as an effective immunogen, and to structurally characterize broadly neutralizing, as well as less potent antibodies, to define their Env epitopes so as to determine the structural basis for their neutralization of HIV-1. This proposal intends to advance research towards a safe and effective, prophylactic HIV vaccine that will save millions of lives. Structural investigations of broadly neutralizing anti-HIV-1 antibodies in complex with the HIV-1 envelope proteins will provide opportunities for design of new immunogens as potential vaccine candidates.